JP2015007077A - Regimens for intra-articular viscosupplementation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide viscosupplementation methods for treating knee joints.SOLUTION: The invention provides viscosupplementation methods for treating osteoarthritis and joint injury with HA-based viscosupplements, particularly viscosupplements with an intra-articular residence half-life shorter than 3 weeks. The viscosupplements contain less than 20 mg/ml HA, at least 5% (w/w) of which is in a gel form such as hylan B. Hylan G-F 20 (Synvisc(R)) is administered in a single intra-articular knee injection of 6±2 ml.

Description

The present invention relates to rheumatology and orthopedics. In particular, the present invention relates to the treatment of cartilage pathology (eg, osteoarthritis) by viscous supplementation.

BACKGROUND OF THE INVENTION Osteoarthritis (OA) is a progressive degenerative disease characterized by degradation of cartilage in the joint, alteration of synovial fluid present in the joint, and subchondral osteosclerosis with osteophyte formation. Patients with OA often present with severe pain that affects many aspects of their daily lives. The prevalence of OA increases with age, and over 60% of OA patients over 60 years of age appear to have some cartilage abnormality (Bjelle (1982) Scand. J. Rheumatol. Suppl., 43: 35-48 (Non-Patent Document 1). OA has become the most expensive form of arthritis, collectively reaching 1-2.5% of the gross national product of the Western nation (Reginster (2002) Rheumatology, 41 (Suppl. 1): 3-6) (Non-patent document 2).

  Synovial fluid lubricates and protects the joint surfaces within the joint. The synovial fluid is mainly composed of high molecular weight polysaccharide hyaluronan (HA, also known as sodium salt of hyaluronic acid, sodium hyaluronate). The concentration of HA in normal human synovial joint fluid is about 3 mg / ml. HA consists of repeating disaccharide units of N-acetylglucosamine and sodium glucuronate (FIG. 1). HA in normal synovial fluid of the joint contains 12,500 disaccharide units with a total molecular weight (MW) of 5 MDa (Balazs et al. (1993) J. Rheumatol. Suppl., 39: 3-9) (Non-Patent Document 3). In OA patients, the concentration of hyaluronan and MW in the synovial fluid are reduced, resulting in a decrease in the synovial fluid's ability to protect cartilage.

  Intra-articular injection of viscoelastic solutions containing high molecular weight HA has been shown to restore normal homeostasis of affected joints. This technique, known as viscosity supplementation, has proven effective in reducing pain and enhancing joint function (eg, Balazs et al. (1993) J. Rheumatol. Suppl., 39: 3-9 (Non-Patent Document 3); Wobig (1998) Clin. Ther., 20 (3): 410-423 (Non-Patent Document 4)).

  Many HA-based viscosity replenishers are available on the market and new products are being developed. Viscous replenishers vary in a number of features including, for example, the source of HA (of animal origin or bacteria), the concentration and MW of HA, and the type and extent of chemical crosslinkers, if any. Usually, most viscous replenishers contain 5-15 mg / ml HA and have a residual half-life of hours to days once injected. Such a viscous replenisher is injected into the knee in a volume of 2-3 ml in a 3-5 injection system every other week. In some cases, pain relief occurs within a few days, continues for weeks, often lasts months, and may even last up to a year. For example, knee viscosity supplementation with Synbisc® (Hylan G-F20; Genzyme Corp., Cambridge, MA) administered 3 times in 2 ml each week is by non-steroidal anti-inflammatory drugs (NSAIDs) + joint cavity puncture It is at least as good as or better than continuous oral therapy over 6 months (Adams et al. (1995) Osteoarthritis and Cartilage, 3: 213-225) (saline solution placebo). Or more effective than the control group of joint cavity puncture (Moreland (1993) Am. Coll. Rheumatol. (57th Ann. Sci. Meeting, Nov. 7-11, San Antonio, TX), 165 (non-patent literature) 6), Wobig (1998) Clin. Ther., 20 (3): 410-423 (Non-patent Document 4)).

  Multiple injection systems have been considered essential for long-term (6 months to 1 year) effects primarily on osteoarthritis pain due to the short half-life of most viscous replenishers (Peyron (1993) J. Rheumatol., 20 (Suppl. 39): 10-15) (Non-Patent Document 7). For example, the intra-articular residual half-life of 1% HA with an average molecular weight of 1.7-2.6 MDa is 11 hours as determined in rabbits. As the molecular weight of HA increases, the residual half-life also increases (eg, 1% Hyran A with an average molecular weight of HA of 6 MDa has a half-life of 1.2 ± 1 day). However, even insoluble gels such as Hiran B containing 0.4% HA have a relatively short residual half-life of 7.7 ± 1 day. Consistent with the half-life data, three 2 ml infusions of SINVISC® into the knee of OA may be significantly more effective in reducing OA pain than two 2 ml infusions. (Scale et al. (1994) Curr. Ther. Res., 55 (3): 220-232) (Non-patent Document 8).

  For treatment of patients with OA in the hip joint with Synbisc®, the recommended dose is a single 2 ml infusion, and if the pain relief is inadequate, the second optional infusion is 1-3 months (Chevalier (2000) Am. Coll. Rheumatol. (64th Annual Scientific Meeting, Oct. 30-Nov. 3, Philadelphia, PA)) (Non-patent Document 9). In hip OA patients, a single intra-articular injection of Synbisc® at 2 ml is significant immediate and up to 3 months (study duration) for symptoms in most of the registered patients Showed the effect. A larger volume of viscous replenisher such as Synbisc® (eg, 4.6 ml or more) is more than equivalent with a few injections compared to multiple injections of 2-3 ml, or a single injection of 2 ml It has not been studied whether it could provide efficacy. As far as is known, the use of larger volumes potentially presents the risk of local side effects such as pain, swelling, and exudation.

  Durolane ™ (Q-Med AB, Uppsala, Sweden) is the only viscous replenisher recommended to be injected at 3 ml at a time. Duroran is an epoxy-crosslinked viscous replenisher with a reported longer half-life (4 weeks) and higher concentrations of HA (20 mg / ml). Increasing the remaining time is believed to reduce the number of injections. Nevertheless, a single infusion of Duroran ™ did not show statistical benefits over placebo (Altman et al. (2004) Osteoarthritis and Cart., 12: 642-649). ).

  Thus, prior to the present invention, it was not known whether a single injection of an HA-based viscosity replenisher, particularly one with a short residence time, could produce the desired long-term therapeutic effect.

  The use of fewer infusions provides clear advantages over multiple infusions, including avoiding side effects, reducing costs, and better patient compliance. There is a continuing need to develop new viscous replacement therapies that provide effective relief for OA patients who do not require multiple injections.

Bjelle (1982) Scand. J. Rheumatol. Suppl., 43: 35-48 Reginster (2002) Rheumatology, 41 (Suppl. 1): 3-6 Balazs et al. (1993) J. Rheumatol. Suppl., 39: 3-9 Wobig (1998) Clin. Ther., 20 (3): 410-423 Adams et al. (1995) Osteoarthritis and Cartilage, 3: 213-225 Moreland (1993) Am. Coll. Rheumatol. (57th Ann. Sci. Meeting, Nov. 7-11, San Antonio, TX), 165 Peyron (1993) J. Rheumatol., 20 (Suppl. 39): 10-15 Scale et al. (1994) Curr. Ther. Res., 55 (3): 220-232 Chevalier (2000) Am. Coll. Rheumatol. (64th Annual Scientific Meeting, Oct. 30-Nov. 3, Philadelphia, PA) Altman et al. (2004) Osteoarthritis and Cart., 12: 642-649

  Methods and compositions are provided for treating joint conditions and reducing the pain and discomfort associated with such conditions. Examples of such pathologies include osteoarthritis and joint damage.

  The present invention, at least in part, provides that a larger volume of a single intra-articular injection of a viscous replenisher provides a long-term therapeutic benefit comparable to that produced by a smaller volume of continuous injection. Based on discovery. In a study conducted in connection with the present invention, one group of knee OA patients received a standard series of injections of Synbisque® 3 times 2 ml into the knee for 3 weeks. The group received a 6 ml single injection under the same conditions. Surprisingly, the efficacy of treatment was found to be similar in both groups, as assessed after 26 weeks of treatment. Thus, a single injection of a larger volume of a viscous replenisher such as Synbisque® can be as effective as several injections of a smaller volume while maintaining favorable safety properties.

Thus, the present invention provides a therapy for intra-articular viscosity replenishment with HA-based viscosity replenishers, particularly those with a residual intra-articular half-life (T _1/2 ) of less than 3 weeks. Viscosity replenishers for use in the method of the present invention further contain less than 20 mg / ml HA (derivatized and / or not derivatized), at least 5% of which is a gel such as eg Hiran B It may be characterized by being in the form of In an illustrative embodiment, the viscous replenisher is Hiran G-F20 (Sinbisk®) containing 8 ± 2 mg / ml HA, with 10% by weight of the HA being in the form of a gel.

  In some embodiments, the viscous replenisher is administered in a single infusion in an amount sufficient to provide a therapeutic effect up to 6 months after the infusion. In some embodiments, the therapeutic effect of a single injection of a larger volume is substantially the same as that achieved by three injections (each 1/3 of the larger volume) administered over the course of treatment. . In an illustrative embodiment, Sinbisque® is administered in a single 6 ml injection rather than 3 2 ml injections over 3 weeks.

  Also provided are methods of administration, compositions and devices for use in the methods of the invention.

  The following summary and the following description do not limit the claimed invention.

FIG. 1 shows the structure of hyaluronan (sodium hyaluronate).

Detailed description of the invention
Definitions The terms “intra-joint half-life”, “residual half-life”, and the terms of their cognate are longer than any of the times applicable to a given viscous replenisher injected into the intra-articular space Refers to time, ie, (a) the time required for 50% clearance of the injected HA gel component, (b) the time required for 50% clearance of the injected HA liquid component, and ( c) The time required for 50% clearance of HA, whether it is liquid, gel, or another form. For purposes of calculating residual half-life, unless otherwise stated, infusion is considered to be administered into the intraarticular space of an adult knee joint. Methods for determining residual half-life are known in the art and exemplary methods are described in the examples.

  The terms “HA liquid”, “HA liquid phase”, “HA liquid component”, “soluble HA”, and terms of their congeners are uncrosslinked or lightly crosslinked having an average molecular weight of less than 20 MDa. Refers to water-soluble HA.

The terms “HA gel”, “HA gel phase”, “HA gel component”, and terms of their congeners do not contain soluble HA or contain HA less than 10% (w / w) soluble HA. Refers to the HA gel that is the water-insoluble part of the composition. Typically, the amount of gel in a given HA-based composition containing a mixture of HA gel and HA liquid can be determined by separating the HA gel from the HA liquid. Separation can be accomplished, for example, by filtering the composition through a 45μ filter that passes through soluble HA and still retains the insoluble phase. In order to maximize the release of soluble HA from the HA gel in a more viscous composition, the composition may need to be diluted with several volumes of solvent that will or will not bring to equilibrium prior to filtration . Further, in general, a pure gel is a preservation (elastic) coefficient (G ′) and a relative (elasticity) coefficient (G ′), which represents the relative degree that the material can recover (elastic response) or flow out (viscous response) as a rate of deformation (test frequency) change It can be distinguished from pure liquids based on the rheological properties of the gel, such as the loss (viscosity) coefficient (G "). Both coefficients are linear functions of frequency. Both coefficients are a function of the polymer solution and gel structure. It proved to be a sensitive probe. Both G ′ and G ″ increase with increasing frequency, but one increases more rapidly than the other. At the point where G '= G ", this frequency is called the crossover frequency (f _c ). The crossover frequency decreases with increasing polymer molecular weight or concentration. In polymer solutions at low frequencies, the elastic stress is relieved. and dominated viscous stress, G "at a frequency below f _c as a result is greater than G '. In contrast, for gels, there is no intersection between G ′ and G ″, and G ′ is greater than G ″ over the frequency range. Unless otherwise specified, the inspection frequency is 0.04-7Hz. For an overview of the physical properties and methods of viscoelastic materials that measure these properties, see, for example, Schulz and Glass, “Polymers as Rheology Modifiers”, ACS Symposium Series 462, 1991, “An Introduction to Rheology”, HA Barnes, JF Hutton. and K. Walters, Elsevier, 1989, and Bohlin Rheometer Application Notes MRK544-01, MRK556-01, and MRK573-01.

  The terms “HA”, “hyaluronate”, “hyaluronan” are used interchangeably and unless otherwise stated, the raw material (bacterial fermented or animal derived), molecular weight, its physical form ( Regardless of example, gel or liquid), or chemical modification (eg, cross-linked or otherwise derivatized) or method of manufacture, refers to any HA.

Therapies The present invention provides methods for viscosity replacement and related methods. According to the present invention, the viscosity replenishment method is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, A single intra-articular injection of a viscous replenisher within 23, 24, 25, 26 weeks consists of administering an amount sufficient to provide a therapeutic effect until 4, 5, or 6 months after the injection. In some embodiments, the therapeutic effect of a single injection of a larger volume is substantially the same as that achieved by three injections (each 1/3 of the larger volume) administered over the course of treatment. is there. In some embodiments, single infusion therapy provides a reduction in joint pain up to 4, 5, or 6 months after infusion.

  The therapeutic effect can be assessed by any suitable method (see, eg, Altman et al. (1996) Osteoarth. Cart., 4: 217-243). For example, the therapeutic effect can be assessed by measuring the reduction in joint pain. The degree of joint pain is a five-point Likert Scale (eg, no, mild, moderate, severe, very severe) or 100 mm visual analog scale as described in the examples Can be classified according to (VAS). Other suitable pain indices include the Health Assessment Questionnaire (HAQ) (Fries et al. (1980) Arthritis Rheumatol., 23: 137-145) and the Arthritis Impact Measurement Scale (AIMS) (Meenan et al. (1980) Arthritis Rheumatol., 23: 146-154).

  The therapeutic effect can also be assessed by measuring an improvement in the degree of functional defect. Functional deficiencies are isolated and validated multidimensional index (SMI) such as Western Ontario and McMaster's Universities (WOMAC ™) OA index for hip and knee OA (Bellamy et al. ( 1988) J. Rheumatol. 34: 1833-1840, see also example), or an aggregated multidimensional index (AMI) such as the Pain Function Index (AFI) for the hip or knee (Lequesne et al. (1987) Scand. J. Rheumatol. Suppl., 65: 85-89).

  The therapeutic effect can also be assessed by comprehensive status assessment by the patient or physician. Global status can be assessed using, for example, a Likert or VAS scale as described in the Examples.

  Additional indices of therapeutic effect include joint examination (eg, Theiler et al. (1994) Osteoarth. Cart., 2: 1-24), exercise capacity based measurements (eg, Rejeski et al. (1995) Osteoarth. Cart., 3: 157-168).

  In some embodiments, the viscosity replenisher is 6 ± 2 ml or more, such as 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7, 7.25, 7.5, to the knee joint. 7.75, administered in an amount of 8ml or more.

Viscosity Supplements HA-based viscosity supplements useful in the methods of the present invention are characterized by any one, any two, or all of the following features. That is,
(I) The viscous replenisher has a residual half-life of less than 3 weeks.
(Ii) The viscous replenisher contains less than 20 mg / ml HA.
(Iii) More than 5% (w / w) of HA in the viscous replenisher is in the form of gel.

  In an exemplary embodiment, the viscous replenisher used in the methods of the present invention is Shinbisque®. Synbisk® is 8 ± in two forms: a soluble form of HIRAN A (average molecular weight 6,000 kDa) and a hydrated gel HIRAN B in physiologically acceptable solutions. Contains 2 mg / ml HA. The Hyran A / Hyran B ratio in Shinbisque® is 9: 1 by weight of HA. Hyran A is a water-soluble hyaluronan that is chemically modified by covalent bonding with a small amount of an aldehyde, typically formaldehyde, whereas Hyran B is Hyran A further crosslinked by divinyl sulfone. Hyran liquor is hydrated Hyran A, a modified form of hyaluronan with a few crosslinks that increase its average molecular weight and increase its viscoelastic properties. Hilangel is a hydrated form of Hiran B and is prepared by cross-linking Hilan A into a continuous polymer network using divinyl sulfone as a bifunctional crosslinker.

  In general, the viscosity replenishers used in the therapy provided by the present invention include shorter than 22 days, such as 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, HA-based viscous replenishers with intra-articular residual half-lives of 9, 8, 7, 6, 5, 4, and 3 days are included. In some embodiments, the residual half-life of the viscous replenisher is greater than 2, 3, 4, 5, 6, or 7 days.

  The viscous replenishers used in the method of the present invention further comprise HA less than 20 mg / ml, for example 1-15, 1-10, 1-5, 5-15, 5-10, 10-15, 6 It may be characterized by containing in the range of -10 and 7-9 mg / ml. The amount of HA in a given composition can be determined in any suitable manner, for example as described in the examples.

  The viscous replenisher composition used in the method of the present invention may further be characterized in that at least 10% by weight of HA in the viscous replenisher is in the form of a gel. For example, in some embodiments, the viscosity replenisher is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, Contains over 70%, 75%, 80%, 85%, 90%, 95%, 99% HA gel. In some embodiments, the ratio of HA gel / HA liquid in the viscous replenisher comprises a range of 1:50 to 10: 1 (w / w), such as 1:50, 1:25, 1:15. , 1:10, 1: 9, 1: 8, 1: 7, 1: 6, 1: 5, 1: 4, 1: 3, 1: 2, 1: 1, 2: 1, 3: 1, 4 : 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, and 10: 1.

  Viscosity replenishers for use in the method of the present invention can further contain water soluble HA in the liquid phase, the molecular weight of HA being in the range of 500-20,000 kDa, for example 500-1,000, 500-1,500. 500-3,000, 500-5,000, 500-7,000, 500-10,000, 500-15,000, 1,000-1,500, 1,000-3,000, 1,000-5,000, 1,000-7,000, 1,000-10,000, 1,000-15,000, 5,000-10,000, and 10,000-15,000 kDa.

  HA can be of animal origin, for example, rooster or umbilical cord, or non-animal, and can be, for example, bacterially fermented. Bacterial fermented HA can be produced as described, for example, in Cooney et al. (1999) Biotechnol. Prog., 15: 898-910. Bacterial fermented HA is also commercially available (eg, Shiseido, Japan, Sigma-Aldrich, USA).

  HA can be derivatized (eg, crosslinked, otherwise modified or stabilized) or cannot be derivatized. Examples of cross-linking agents include aldehydes, epoxides, polyazilyls, glycidyl ethers (eg, 1,4-butanediol diglycidyl ether), and divinyl sulfone.

  Specific examples of viscosity replenishers useful in the methods of the present invention include Adant ™, Arthrase ™, Arthrum ™, Fermathron ™, Goon (TM), Hyalart (TM) / Hyalgan (TM), Hy-GAG (TM), Hyaject (TM), Hyalbrix (Hyalubrix) Trademark), NeoVisc ™, Supartz ™ / Artz ™, Synvisc ™, Orthovisc ™, Ostenil ™ ), Sinovial (TM), Suplasyn (TM), and Synochrom (TM), Viscorneal (TM) (e.g., Lens Desk Reference (Physicians' Desk Reference) (TM), see 2004). Other products suitable for the method of the present invention are described in U.S. Patent Nos. 5,143,724, 4,713,448, 5,099,013, 5,399,351, 6,521,223, 5,827,937, U.S. Patent Application No. 60 / 533,429. A viscous replenisher.

  The preparation of viscous replenishers comprising hyran and hyran A and hylan B is described, for example, in US Pat. Nos. 5,143,724, 4,713,448, 5,099,013, and 5,399,351.

  In some embodiments, the viscosity replenisher is 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40. , 41, 42, 43, 44, 45, 46, 47, 48, 49, or Durolane ™ and / or other viscous replenishers with a residual half-life longer than 50 days.

  Viscosity replenishers may also contain additional active or inactive ingredients, including non-steroids such as, for example, Ibuprofen ™, Diclofenac ™, and Piroxicam ™. Anti-inflammatory drugs (NSAIDs), for example anesthetics such as Lidocaine ™ and Bupivacaine ™, opioid analgesics such as codeine and morphine, corticosteroids such as dexamethasone and prednisone, methotrexate Includes antineoplastic agents such as (Methotrexate (TM), 5-fluorouracil and Paclitaxel (TM), and antiviral agents such as, for example, Acyclovir (TM) and Vidarabine (TM) . Viscous supplements may also contain components such as cells (eg, chondrocytes or mesenchymal stem cells), proteins, DNA, vitamins or other desirable biologically active materials.

Uses and Administrations The present invention provides methods and compositions for treating a subject having a joint condition and reducing the pain and discomfort associated with such condition. Examples of such pathologies include osteoarthritis (primary (idiopathic) or secondary), rheumatoid arthritis, joint damage (eg, repetitive movement damage), cartilage pathology (cartilage softening), and arthritis The previous state is included. The present invention further provides methods for reducing the pain associated with such pathologies. The method can be performed in a human or non-human subject in need of treatment for a joint condition.

  Examples of administration sites include knee, shoulder, chin and carpal joints, elbows, hips, wrists, feet, and joints between lumbar joint processes (intervertebral) in the spine. The volume injected into any of these joints will be at least twice the currently recommended dose for that joint.

  The present invention further comprises 6 ± 2 ml of Synbisc®, for example 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7, 7.25, 7.5, 7.75, 8, 8.25, 8.5, 8.75, 9, 9.25, 9.5, 9.75, providing a viscous replenishment device comprising a pre-filled single use syringe having a single unit dose of 10 ml or more. While it is preferred to use one syringe provided herein to provide a single dose, two or more syringes may be provided for the required dose. For example, a single dose of 6 ml can be achieved using 3 syringes containing 2 ml each.

  The following examples provide illustrative aspects. The examples do not limit the invention in any way. Those skilled in the art will recognize numerous modifications and variations that may be implemented within the scope of the present invention. Such modifications and variations are therefore encompassed by the invention.

Example 1: Intra-articular injection of Synbisc® in patients with OA A prospective open-label study was conducted, outpatients with symptomatic tibial-femoral OA (ranging from 50-80 for a 100 mm VAS score) Safety of Synbisc® 4 ml or 6 ml intra-articular injection versus current dosing regimen of 2 ml of 3 ml of Synbisque® in the study knee (comprehensive OA pain) And efficacy (including duration of action) was evaluated. Other conditions included are ages 40 and older, Kellgren-Lawrence grades II-III within the last 3 months, tense effusioin, mechanical impairment, or recent (less than 2 years) There was no trauma. 100 patients (mean age 61 years in the range 59-66 years, 55% women) were randomized into 5 groups. That is,
One injection of group 1-6ml,
One injection of group 2-4ml,
Group 3-2 injections of 4 ml every 2 weeks,
Standard therapy with 3 4 ml infusions every group 4-1 weeks and 3 2 ml infusions after group 5-1 weeks.

  The patient was then followed up to 6 months (2 weeks, 3 weeks, 8 weeks, 16 weeks, and 24 weeks). The primary and secondary endpoints used are listed below.

A. Patient self-assessment of OA pain The primary efficacy endpoint of this study is to evaluate the effectiveness of viscosity supplementation with Synbisc® in patients with knee OA with respect to the relief of OA pain in the study knee It is. This was measured for 100-mm VAS self-assessed patients with a painless (0 mm) to extremely painful (100 mm) endpoint within the last 48 hours and was performed 24 weeks after the first infusion.

B. Comprehensive patient self-assessment Patients should be aware of 100-mmVAS ranging from very good (0mm) to very poor (100mm), taking into account all relevant signs and symptoms over the previous 48 hours The overall condition of their target knees was ranked. The actual explanation presented to the patient was as follows. That is, "Use the vertical line below to indicate the general general condition of your (study) knee at this visit." A score of “0” on the left or “0” indicates “very good”, whereas a score of “100” indicates “not very good”.

C. Warmac (trademark)
The patient implemented the VAS version of Warmac ™ described in Bellamy et al. (1988) J. Rheumatol., 15 (12): 1833-40. This scale is a three-dimensional, disease-specific, self-administered, health measure. It detects clinically important patient-related symptoms in pain area, stiffness, and physical function in a total of 24 questions. Warmac ™ was provided to patients in local language and usually finished in less than 5 minutes. The subsections of Warmac ™ are as follows:

Warmac ™ Section A consisted of questions about the level of pain during activity and scored responses (by the patient) with VAS ranging from painless (0 mm) to extremely painful (100 mm). Pain was evaluated for the following scenario. That is,
1. When walking on a plane? [When walking]
2. When going up and down stairs? [When going up the stairs]
3. When to sleep at night? [Night onset]
4). When to sit or lie down? [At rest]
5. When is it upright? [When supporting weight]
It is. The average minor score for Warmac section A was based on the response to each section A component.

Warmac ™ Part B (stiffness score) consists of questions about the severity of stiffness during activity, with a (patient) response in a VAS ranging from no stiffness (0mm) to extreme stiffness (100mm). Scoring. Stiffness was evaluated for the following scenarios. That is,
1. After walking in the morning? [Morning stiffness]
2. Is it relatively late in the day, at rest? [Stiffness occurs relatively late in the day]
Met. The average minor score for Section B was based on the response to each component of Section B.

Warmac ™ Section C consisted of questions about dysfunction in action and scored responses (by the patient) in VAS ranging from not difficult (0 mm) to extremely difficult (100 mm). The functional impairment was evaluated for the following scenarios. That is,
1. Can you go down the stairs? [Morning stiffness]
2. Can you go up the stairs? [Stiffness occurs relatively late in the day]
3. Can you get up from the sitting position? [Stand up]
4). Do you stand up? [Stand up]
5. Can you flex to the floor? [Can be bent]
6). Can you walk on the plane? [I can walk the plane]
7). Can I get on and off the car? [car]
8). Can you go shopping? [shopping]
9. Can I wear socks / stockings [Remove socks / stockings]
10. Can I lie down in bed [I can lie down in bed]
11. Can I get in and out of the bathtub [I can enter and leave the bathtub]
12 Can I sit down?
13. Can I enter and exit the toilet [Can enter and exit the toilet]
14 Can you do heavy housework [Can do heavy housework]
15. Can you do mild housework? [Can do mild housework]
Met. The average minor score for Section C was based on the response to each component of Section C.

  The change from baseline in the total Warmac ™ score from the three Warmac ™ sections (A, B and C) at all time points after the first injection was used as a secondary endpoint. analyzed.

D. Physician OA Comprehensive Assessment After the patient has completed the Comprehensive Assessment and WOMAC (TM), the investigator will investigate the 100-mmVAS ranging from very good (0mm) to very poor (100mm) Rated the overall condition of the knee. This assessment was based on the patient's disease symptoms, functional potential and physician examination. The doctor determines the general condition of the patient's knee at this visit to the left (“0”) for the “very good” line and to the right for “very bad” (“ 100 ") was indicated using the line represented.

E. Results Table 1 shows the results showing a reduction in pain (VAS; 0 corresponding to no pain within the last 48 hours and 100 corresponding to extremely painful) 24 weeks after the first infusion compared to baseline. Pain was reduced by 34.9 mm in the 1 × 6 ml group compared to 36.7 mm in the 3 × 2 ml group that scored best. In the group treated with 1 × 4 ml or 2 × 4 ml, this decrease was less dramatic (only a 24 mm decrease).
(Table 1) Patient self-assessment of OA pain (assessment from baseline)

（表３）患者の包括評価（ベースラインからの変化）

（表４）医師の包括評価（ベースラインからの変化）

（表５）治療群の格付け

Pain, stiffness and dysfunction as measured by Western Ontario and McMaster Universities Osteoarthritis Index (Warmack ™) (Table 2 for Warmac ™ A) Secondary efficacy endpoints including improvements in patients, patients (Table 3) and physician (Table 4) comprehensive knee OA assessment showed the same trend. The treatment groups were ranked in order of effectiveness and the results are shown in Table 5. The extension of the duration of the effect observed in group 1 was surprising. Regarding safety, there were reports of minimal or moderate strength local knee side effects (pain, swelling or exudation) in 10% of patients in each group (1 × 6 ml and 3 × 2 ml).
Table 2: Warmac ™ A pain score (change from baseline)

(Table 3) Comprehensive evaluation of patients (change from baseline)

(Table 4) Comprehensive evaluation of doctors (change from baseline)

(Table 5) Rating of treatment groups

  No significant differences were observed between treatment groups in terms of safety, with Group 1 (1 x 6 ml) generally having only minimal side effects. These results also suggest that a volume of SINVISC® greater than 2 ml can be safely administered to reduce pain in patients with knee osteoarthritis.

Example 2: Determination of the residual half-life of Sinbisque®
A. Incorporation of ¹⁴ C-acetate into rooster and scab organ culture hyaluronan Young roosters (3-6 months old) were sacrificed by cervical dislocation. Rooster crests were completely cleaned with (80%) ethanol and then excised at the base using a scalpel. Excess blood was squeezed out of the scab, and the scab was placed into a sterilized saline solution, transferred to a laminar flow hood, and rinsed in an additional 3 volumes of sterilized saline solution. The crest was then incised along the midline of the blood vessel and a rectangular segment of pink skin tissue was excised. Slice the tissue into thin sections with a scalpel, Ventrex medium HL-1 (Ventrex Labs), 5 mg / ml testosterone propionate (Belmar Laboratories, Inwood, NY), ¹⁴ C acetic acid 20 μCi / ml (ICN Radiochemicals, Irvine, CA, 1 mCi / ml), 0.1 mg / ml penicillin, 0.1 mg / ml streptomycin and 0.1 mg / ml fungizone (Hazelton, Lenexa, KS). Individual cultures were prepared in 60 mm plastic petri dishes and contained about 1.5 g of tissue and 15 ml of medium. After incubating the culture for 72 hours in a 5% CO ₂ environment, the tissue was separated from the medium by centrifuging at 10,000 g for 10 minutes. Tissue pellets were frozen in 30 mm Petri dishes. Typically, radiolabeled frozen scabs and tissues were stored frozen for 1 to 72 hours prior to continuing processing to prepare lanthanum.

B. Preparation of Radiolabeled Hyran Solution Hyran A was prepared as follows. Frozen and radiolabeled sections of tissue were placed in a reaction medium containing acetone, formalin (37% formaldehyde solution), chloroform, and sodium acetate at a ratio of 0.75 g tissue per gram of reaction medium. After the reaction was allowed to proceed for 18-20 hours, tissue sections were collected, washed 3 times with acetone, and then dried in a laminar flow hood. Next, 4 volumes of distilled water were added to the dried tissue section to extract radiolabeled lanthanum. After this aqueous extraction was performed at 4-6 ° C., the aqueous extract was extracted and the same amount of water was added again for secondary extraction. Solid sodium acetate was dissolved in the aqueous extract to a concentration of 1% and hydran fibers were precipitated by slow addition to 4 volumes of 95% ethanol. Radiolabeled hydran fibers were washed twice with acetone and stored cold under acetone.

  4 in 4 ml of pooled, labeled, pyrogen-free, phosphate buffered saline (Biotrics Inc., Ridgefield, NJ, lot 122-1) Dissolved by slow rotary mixing at 3 ° C for 3 days. After complete dissolution, the radioactive hydran solution was diluted 5-fold with unlabeled hydran solution. Rotational mixing of the mixture at 4 ° C. was maintained for an additional 5 days.

C. Preparation of radiolabeled gel Tritium-labeled water (New England Nuclear, 100 mCi / ml) was mixed into the reaction mixture used to crosslink hydran liquid (Hillan A) to Hilangel (Hillan B). The cross-linking reaction was performed as follows. Hyran A fibers were swollen for about 3 hours in tritium-labeled water. Concentrated sodium hydroxide was added and the mixture was stirred vigorously until the solution was homogeneous (about 15 minutes). Divinyl sulfone was diluted in water to a concentration of 50% and added to the reaction mixture with vigorous stirring. The reaction mixture was left at room temperature (22 ° C.) for an additional 55 minutes, during which time the polysaccharide chains were crosslinked with divinyl sulfone into a continuous polymer gel (Hilanger). By performing this reaction in tritium-labeled water, tritium is covalently bonded to carbon within the divinylsulfonyl bridge. To lower the pH below 12, the reaction was terminated by the addition of 10 volumes of sterile, pyrogen-free saline solution. Saline washing also caused the angel to swell to its equilibrated hydration. The angel was washed with a saline solution to remove unreacted divinylsulfone, unreacted tritium, and other reactive products, and the pH was lowered to 7. After the excess salt solution was separated from the gel by filtration, the gel was passed through a 25 g needle 5 times to decompose the solid gel into a form that could be easily injected. In this form, the tritium labeled gel was exhaustively dialyzed against a sterile, pyrogen-free saline solution to remove any non-covalently bound tritium.

D. Preparation of Hilangel-Hillan Liquid Mixture Tritium-labeled Hilangel (3.04 g) was added directly to 11.63 g of ¹⁴ C-Hyran liquid and the mixture was placed in a Glen Mills mixer for 48 hours. The mixture was then passed 10 times through 18 g, 21 g, and 25 g needles to ensure homogeneity and ease of injection.

E. Measurement of HA concentration and amount of radiolabel The concentration of hyran polysaccharide in the gel and liquid components of the mixture was determined by the automated carbazole method, and its repeating glucuronic acid monomer (3) was assayed (2.07) And the remainder of the hydran polysaccharide chain was counted. Prior to the determination of glucuronic acid, the angel was hydrolyzed and hydrolyzed by adding a weighed 0.1 g sample of gel to 0.2 ml of 1N H ₂ SO ₄ in a tightly capped screw-cap tube. Was allowed to proceed for 2 hours at 100 ° C. Samples completely solubilized by this procedure were neutralized by 0.2 ml 1N NaOH by carbazole procedure prior to analysis of HA.

  The carbazole approach involves measuring the amount of hexuronic acid (glucuronic acid) in a sample. A method for determining hexuronic acid concentration by a colorimetric method was reported by Dische et al. (1947) J. Biol. Chem., 167: 189-198. The method is based on the dye reaction of hexuronic acid with sulfuric acid and carbazole. An updated automated method for the determination of hexuronic acid was reported by Balazs et al. (1965) Anal. Biochem., 12: 547-558. The sample is heated in a sulfuric acid / borate medium and reacted with carbazole. Carbazole reacts with hexuronic acid to form a pink complex with an absorption maximum at 530 nm. For automated methods, samples and standards are aspirated through a continuous flow analyzer using a peristaltic pump. Reagents (acid and carbazole) are added, heated in the reaction chamber, and absorbance is read at 530 nm with a continuous flow colorimeter.

  The amount of radioactivity of the test article was determined by scintillation counting in an ISOCAP 300 liquid scintillation counter (Nuclear Chicago) using Scintiverse Bio HP (Fisher Scientific) as a scintillation agent. Raw CPM data was converted to DPM using the ISOCAP 300 external standard ratio program and normalized to tritium liquid scintillation quenching standards or carbon-14 liquid scintillation quenching standards (Amergham, Arlington Heights, II).

F. Determination of Shinbisque® Residual Half-Life Clearance of Synbisque® and its gel and liquid components from the knee joint was determined in New Zealand white rabbits weighing 2.5-3.5 kg. Rabbits were sacrificed at 24 hours, 3, 7, and 28 days, respectively. Radioactive material prepared essentially as described above was administered as a 0.3 ml intra-articular injection (0.086 ml / kg body weight). This dose level is expected to be equivalent to a single 6 ml dose of Synbisc® to a 70 kg human. The corresponding amount to be administered to other animals is also directly proportional to the animal's body weight.

  DPM was obtained for each tissue as outlined above and, where appropriate, DPM / mg was calculated directly. Both total DPM recovered and DPM and DPM / mg for each joint tissue were calculated individually for each animal. These values were then averaged for each time point and expressed as mean ± standard error of the mean. Reported averages were the smallest and most significant two-digit numbers, even when the values were small and the animal-to-animal variability was large. The total mean DPM recovered from the joint at each time point was calculated by averaging the sum of the individual animals.

Half-life determinations were made by fitting the average for each time point to an exponential function (Y = Ae ^kx ). The approximate standard error was obtained from the curve fit and the expected percent error was obtained by dividing by A. This was multiplied by the half-life to obtain the expected error in half-life.

G. Results The gel component (Hyran B) of Synbisc® is part of a longer half-life. Based on the clearance of the radioactive material, the residual half-life of the gel component was determined to be 7.7-8.8 days. Thus, by day 30, more than 95% of the gel will be eliminated. A theoretical calculation based on the experimentally determined half-life of the gel was performed to approximate the amount of gel expected to be present in the human joint after a single 6 ml injection. Assuming about 6 mg of gel was injected into the knee of a human subject, 21 days after injection, the amount of gel remaining would be about 0.9 mg.

  The liquid component (Hyran A) of Synbisc® is eliminated more quickly than the gel component. The half-life of the liquid component was determined to be 1.2-1.5 days. By day 7, 99% of the injected material had been removed from the rabbit knee joint.

  A rabbit muscle implant study was also conducted. Microscopic examination at 7 and 30 days after embedding did not detect any residual test material, which is consistent with the intra-articular clearance study.

  The specification is most thoroughly understood in light of the teachings of the references cited within the specification. Aspects in the specification are not to be construed as limiting the scope of the invention to the exemplification of aspects of the invention. Those skilled in the art will readily recognize that many other embodiments are encompassed by the present invention. All publications and patents cited in this disclosure are incorporated by reference in their entirety. Any material incorporated by reference that is inconsistent or inconsistent with this specification will be replaced by this specification. Citation of any reference herein is not an admission that such reference is prior art to the present invention.

  Unless otherwise stated, all numbers representing amounts of ingredients, cell cultures, treatment conditions, etc. used in the specification, including the claims, are understood to be modified in all cases by the word “about”. It should be. Thus, unless stated otherwise, the numerical factors are approximate and can vary depending on the desired properties sought to be obtained by the present invention. Unless otherwise stated, the word “at least” preceding a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize many equivalents to the specific embodiments of the invention described herein or will be able to ascertain using no more than routine experimentation. Such equivalents are intended to be encompassed by the above claims.

Claims (35)

  A viscosity replenishment method for treating the knee joint of a subject, comprising a single intra-articular administration of 4 ml or more.   2. The method of claim 1, wherein the viscous replenisher contains less than 20 mg / ml HA.   2. The method of claim 1, wherein the subject is a human.   4. The method according to claim 3, wherein the total volume of the viscous replenisher in a single administration is 6 ± 2 ml or more.   5. A method according to claim 4, wherein the amount is 6 ml.   2. The method of claim 1, wherein the viscous replenisher is administered to a subject in need of treatment for joint pain.   2. The method of claim 1, wherein a single administration of the viscous replenisher is therapeutically effective for at least 4 months. The method of claim 1, wherein the viscous replenisher is characterized by one or more of the following characteristics:
(I) the viscous replenisher has a residual half-life of less than 3 weeks;
(Ii) the viscous replenisher contains less than 20 mg / ml HA,
(Iii) More than 5% (w / w) of HA in the viscous replenisher is in gel form.   9. The method of claim 8, wherein the residual half-life is greater than 3 days.   2. The method of claim 1, wherein the viscous replenisher contains 8 ± 2 mg / ml HA.   2. The method according to claim 1, wherein the HA in the viscous replenisher is derived from an animal.   12. The method according to claim 11, wherein the viscous replenisher is produced from rooster scabbard.   2. The method of claim 1, wherein the HA in the viscous replenisher is derived from bacteria.   The method of claim 1, wherein at least a fraction of the viscous replenisher is crosslinked.   15. A method according to claim 14, wherein the cross-linking agent is formaldehyde.   The method of claim 15, wherein the viscous replenisher comprises Hilan A.   17. The method according to claim 16, wherein the average molecular weight of HA in Hiran A is 6,000 kDa.   15. A method according to claim 14, wherein the cross-linking agent is divinyl sulfone.   The method of claim 18, wherein the viscous replenisher comprises hydran B. 15. A method according to claim 14, wherein the viscous replenisher comprises Hiran A and Hiran B.   21. The method of claim 20, wherein the ratio of Hiran A / Hilan B is 9: 1 by weight of HA.   The method according to claim 1, wherein the viscous replenisher contains 8 ± 2 mg / ml HA, 10% by weight of which is in the form of a gel.   The method of claim 1, wherein the viscous replenisher is Synvisc®.   24. The method of claim 23, wherein the viscous replenisher further comprises a component selected from the group consisting of a non-steroidal anti-inflammatory agent, an anesthetic, an opioid analgesic, a corticosteroid, an antineoplastic agent, and an antiviral agent. .   A viscosity replenishment method for treating a knee joint in a human subject, comprising a single intra-articular administration of 6 ± 2 ml of Sinbisque®.   26. The method of claim 25, wherein the amount administered is 6 ml.   9. A method for treating a joint condition comprising the viscosity replenishment method of claim 1.   28. The method of claim 27, wherein the joint condition is associated with osteoarthritis.   A method of treating osteoarthritis pain in a knee joint of a human subject comprising administering a single intra-articular injection of 6 ± 2 ml of Sinbisque®.   32. The method of claim 31, wherein the amount is 6 ml.   A viscous replenishment device comprising a syringe containing 4 ml or more of Shinbisque®.   32. The device of claim 31, wherein the device is sterilized.   32. The device of claim 31, wherein the syringe contains 6 ± 2 ml of Synbisque®.   32. The device of claim 31, wherein the amount of Synbisc® in the syringe is 6 ml.   The sybisk® further comprises a component selected from the group consisting of non-steroidal anti-inflammatory agents, anesthetics, opioid analgesics, corticosteroids, antineoplastic agents, antiviral agents, and cells. 31. The device according to 31.

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